PROCESS FOR THE PREPARATION OF PHENYLALANINE DERIVATIVES OR HOMOLOGUES CONTAINING A GUANIDINO OR MODIFIED GUANIDINO GROUP USEFUL FOR THE PREPARATION GnRH OF ANTAGONISTIC PEPTIDES

Abstract: Methods of making unnatural amino acids which can be incorporated into peptides which either inhibit or promote the secretion of gonadotropins by the pituitary gland and inhibit the release of steroids by the gonads which method comprises subjecting an alpha -amino acid having fomula (i), wherein j is 1, 2 or 3, to nitration conditions by forming a reaction mixture which includes said amino acid, concentrated sulfuric acid and concentrated nitric acid having at least about 2 moles of HNO3 for each mole of alpha -amino acid and maintaining said reaction mixture at a temperature of about 5 DEG C or below to cause said nitration to occur predominantly at the 4-position, reacting said 4NO2-substituted product with an appropriate reagent to add an amino-protecting group to said alpha -amino acid, treating said protected amino acid to hydrogenate said substituted nitro moiety, and transform it to an amino moiety, and dissolving said hydrogenated amino acid in a suitable solvent and reacting it with diphenylcyanocarbonimidate to form a cyanoguanidino intermediate which is reactive with a nucleophylic moiety to produce a desired unnatural amino acid having formula (a) or formula (b).

Description: PROCESS FOR THE PREPARATION OF PHENYLALANINE DERIVATIVES OR HOMOLOGUES CONTAINING A GUANIDINO OR MODIFIED GUANIDINO GROUP USEFUL FOR THE PREPARATION GnRH OF ANTAGONISTIC PEPTIDES This invention relates to the preparation of new unnatural amino acids, which are derived from diamino acids, such as NH 2 Phe, which amino acids are useful in the 5 synthesis of biologically active peptides. More particularly, it relates to preparing peptides having such unnatural amino acids, which peptides are prepared by forming the protected unnatural amino acid and then incorporating it into such a peptide as a part of a 10 standard chain elongation synthesis process. These unnatural amino acids are particularly useful for forming GnRH antagonists which inhibit gonadal function and the release of the steroidal hormones, progesterone and testosterone and also to GnRH agonist 15 peptides which promote the release of such steroids. BACKGROUND OF THE INVENTION The pituitary gland is attached by a stalk to the region in the base of the brain known as the 20 hypothala us. In particular, follicle stimulating hormone (FSH) and luteinizing hormone (LH) , sometimes referred to as gonadotropins or gonadotropic hormones, are released by the pituitary gland. These hormones, in combination, regulate the functioning of the gonads to 25 produce testosterone in the testes and progesterone and estrogen in the ovaries, and they also regulate the production and maturation of gametes. The release of a hormone by the anterior lobe of the pituitary gland usually requires a prior release 30 of another class of hormones produced by the hypothalamus. One of the hypothalamic hormones acts as a factor that triggers the release of the gonadotropic hormones, particularly LH, and this hormone is referred to herein as GnRH although it has also been referred to as LH-RH and as LRF. GnRH was isolated and characterized as a decapeptide some 20 years ago, and it was found that analogs of GnRH having a D-isomer instead of Gly in the 6-position, such as [D-Ala 6 ]-GnRH (U.S. Patent No. 4,072,668) having the following formula: pGlu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-NH 2 , have greater binding strength to the receptor and greater biological potency than the native hormone both in vitro and in vivo. Peptides are compounds which contain two or more amino acids in which the carboxyl group of one acid is linked to the amino group of the other acid. The formula for GnRH, as represented above, is in accordance with conventional representation of peptides where the amino terminus appears to the left and the carboxyl terminus to the right. The position of the amino acid residue is identified by numbering the amino acid residues from left to right. In the case of GnRH, the hydroxyl portion of the carboxyl group of glycine at the C-terminus has been replaced with an amino group(NH 2 ) i.e. the C-terminus is amidated. The abbreviations for the individual amino acid residues above are conventional and are based on the trivial name of the amino acid, e.g. pGlu is pyroglutamic acid, Glu is glutamic acid, His is histidine, Trp is tryptophan, Ser is serine, Tyr is tyrosine, Gly is glycine, Leu is leucine, Nle is norleucine, Orn is ornithine, Arg is arginine, Har is homoarginine, Pro is proline, Sar is εarcosine, Phe is phenylalanine, Ala is alanine, Val is valine, Nva is norvaline, lie is isoleucine, Thr is threonine, Lys is lysine, Asp is aspartic acid, Asn is asparagine, Gin is glutamine, and Met is methionine. Except for glycine, the recited amino acids should be understood to be of the L-configuration unless noted otherwise. There are reasons for desiring to prevent ovulation in female mammalians, and the administration of GnRH analogs that are antagonistic to the normal function of GnRH have been used to suppress or delay ovulation. For this reason, analogs of GnRH which are antagonistic to GnRH are being investigated for their potential use as a contraceptive or for regulating conception periods. GnRH antagonists may also be used for the treatment of precocious puberty and endo etriosis. Such antagonists have also been found useful to regulate the secretion of gonadotropins in male mammals and can be employed to arrest spermatogenesis, e.g. as male contraceptives for treatment of male sex offenders, and for treatment of prostatic hypertrophy. More specifically, GnRH antagonists can be used to treat steroid-dependent tumors, such as prostatic and mammary tumors, and for the control of the timing of ovulation for in vitro fertilization. In the female, they can also be used to treat hirsutism, endometriosis, premenstrual syndrome (PMS) , etc. On the other hand, GnRH agonists function in the same manner as GnRH in promoting the release of LH and FSH, and agonists which exhibit greater biopotency and/or longer duration of action are considered valuable. Of particular interest are methods for making unnatural amino acids which enhance biological activity as a result of their substitution into peptide analogs. It is also desired to provide methods for making improved peptides by a chain elongation process which either are strongly antagonistic to endogenous GnRH and prevent secretion of LH and FSH and the release of steroids by the gonads of mammals or are strong agonists of GnRH. SϋMMARY OF THE INVENTION The present invention provides methods for making unnatural amino acids and for preparing peptides using such previously prepared unnatural amino acids, α- amino-protected amino acids are synthesized having a side chain which contains a modified guanidino group or a guanidino equivalent or a derivative that is obtained by further elucidation of a modified guanidino group, as set forth hereinafter, and which most preferably contains a triazole or substituted triazole group. These amino acids are useful in preparing peptides which inhibit the release of gonadotropins in mammalians, including humans, and also in preparing improved GnRH analogs which are strong agonists of GnRH and can be used to promote the reproduction processes of mammalians. The invention provides methods for making protected unnatural amino acids having the following formula U * : N-CN HO or a heterocyclic rearrangement thereof, where X 1 is an - amino protecting group; j is 1,2 or 3; R, is H, CH 3 or CH 2 CH 3 ; R 2 is alkyl (C 1 to C 6 ) , cyclohexyl, or -(CH 2 ) p -R 3 ; wherein p is an integer between 0 and 6 and R 3 is methyl, ethyl, phenyl, pyridinyl, pyri idinyl, purinyl, imidazolyl, indolyl, naphthyl, NHR 4 or OH; and R 4 is H or CH 3 . Preferably the method results in an unnatural amino acid has one of the following formulas including any heterocyclic rearrangement which occurs: (a) where X 1 is an α-amino protecting group; j is an integer from 1 to 3; R, is H, CH 3 or CH 2 CH 3 ; and R 2 is alkyl (C- to c β i cyclohexyl, naphthyl, pyridyl, pyrimidyl, pyrazinyl, indolyl, quinolinyl or imidazolyl; (b) NHR 6 / 0 N - C II r-~ \ II II HO-C-CH-(CH,) , -( )-N-C N I l ^/ I \ / (X 1 )NH H NR 5 where X 1 is an α-amino protecting group; j is an integer from 1 to 3; R 5 is H, methyl or ethyl; and R 6 is H or acyl(C, to C 6 ) ; (c) NH π where X 1 is an α-amino protecting group; j is an integer from 1 to 3; M t is NR 7 or NH-(CH 2 ) -R 8 wherein p is an integer from 0 to 3, R 7 is methyl, ethyl, propyl or phenyl; R g is O or desR g ; and R, is H or alkyl (C, to C 6 ) ; and ( d ) NH where X 1 is an α-amino protecting group; j is an integer from 1 to 3; M 1 is NR 7 or NH-(CH 2 ) wherein p is an integer from 0 to 3, and R 7 is methyl, ethyl, propyl or phenyl. More preferably, the methods are carried out to make amino acids with the formula: NH 2 N - C HO where X 1 and p are is as previously defined. Such amino acids are valuable in both L- and D-isomer forms to synthesize GnRH analogs and a wide variety of other peptides by the standard chain elongation methods. Modification of a primary amino function of an initially amino- odified Phe or a ho olog thereof is carried out by treatment of the appropriately protected amino acid with appropriate reagents. These amino acids are broadly referred to as cyanoguanidines and are prepared by reaction of an amino group with diphenyl cyanocarbonimidate (I) : N-CN N-CN II II Q-NH 2 + PhO-C-OPh > Q-NH-C-OPh (I) (ID wherein "Q" is used to broadly represent the major portion of an amino acid having a side ' chain primary amino group, such as the amino acid which is depicted above, as a part of formula U * , which amino acid would have its α-amino group appropriately protected. The amino acid having the N-substituted-N'-cyano-O-phenylisourea moiety (II) is then further functionalized by reaction with a second nucleophile HXR 2 to produce cyanoguanidine-containing amino acids having the formula (III) : N-CN N-CN Q-NH-C-OPh + HXR Q-NH-C-XR (ID (III) For example, N-CN N-CN (aminomethyl cyanoguanidino Q-NH-C-OPh + NH 2 CH 3 -> Q-NH-C-NHCH 3 moiety) (II) (III) For example , where HXR = H 2 N-CH 2 -pyridine , the result is : N-CN (3 aminomethyl II pyridyl Q-NH-C-HNCH 2 cyanoguanidino moiety) This group may also be referred to (IUPAC nomenclature) as N-g-cyano-N-g'-3-methylpyridylguanidino. Such compounds can be hydrolyzed under acidic conditions to create amino acids that can also be used to produce compounds which are also biopotent—for example: O II N-CN N-C-NH 2, || TFA/H 2 0 || Q-NH-C-NHCH 3 > Q-NH-C-NHCH 3 22 ° C Cyclic moieties result when -XR contains a second nucleophilic site and X has the general form: M..-(CH 2 ) p -M 2 where M 1 is NR 1 and M 2 is NH, 0 or NR", with p being 0,1,2 or 3. Examples of such nucleophiles include H 2 NNH 2 , CH 3 HNNH 2 , CH 3 HNNHCH 3 , H 2 NOH, and H 2 N-CH 2 -CH 2 OH. In this case, the cyanoguanidine moiety that is formed is converted into the corresponding heterocycle (V) which forms from the initial intermediate by reaction of the omega amino group with the cyano group such as: Q-NH-C-M (CH 2 ) p -M 2 > Q-NH-C-M,-(CH 2 ) p • (V) For example, where -XR 2 = -HNNH 2 , NH 2 / N-CN N—C (1,2,4 triazole moiety) Q-NH-C-HNNH 2 > Q-NH-C N N H Furthermore , where -XR = -CH 3 NNHCH 3 NH O II π N-CN N - C N - C II II I H * II I Q-NH-C-CH 3 NNHCH 3 — > Q-NH-C N-CH 3 > QNH-C NCH 3 N N I I CH 3 CH 3 which may then undergo hydrolysis, as indicated above. Generally, these unnatural amino acids are used to synthesize peptides which are antagonists or agonists of GnRH, i.e., they either strongly inhibit the secretion of gonadotropins by the pituitary gland of mammalians, including humans, or they strongly promote such secretion or release. These peptides are analogs of GnRH containing one or more unnatural amino acids of the formula U * in the 3-position, the 5-position, the 6-position and/or the 8-position. When U * is in the 3- and/or 6-position, it is always in the form of a D- isomer; whereas when U * is in the 5- and/or 8-position, it is always in the form of an L-isomer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS As previously mentioned, the unnatural amino acids (which can be L- or D-isomers) are represented by the formula U *: O N-CN H HOO--CC--CCHH--((CCHH P2 )) j ,(Uj)>VNNHH j2 -- -NNN--C-NR, (X 1 )NH H R, wherein X 1 , j, R.. and R 2 are as defined previously. The α-amino group is substituted by a suitable protecting group, e.g. Boc, to permit its direct use as a part of a chain-elongation method for synthesizing a peptide beginning at the C-terminus. In each of the exemplary peptides formed using amino acids made by the invention, there is at least one D-isomer. More specifically, biopotent GnRH antagonists are represented by the following Formula (F.,) : G-AA^(A)D-Phe-AA 3 -Ser-AA 5 -AA 6 -AA 7 -AA 8 -Pro-AA 10 wherein G is hydrogen or an acyl group having 7 or less carbon atoms; AA 1 is dehydroPro, D-pGlu, (A)D-Phe, (B)D-Trp, Pro, or / Θ-D-NAL; A is H, Cl, F, N0 2 , CH 3 , OCH 3 , C β Me/4Cl, Cl 2 or Br; B is H, N0 2 , NH 2 , OCH 3 , F, Cl, Br, CH 3 , N in For or N in Ac; AA 3 is U * , D-PAL, /3-D-NAL or (B)D-Trp; AA 5 is U * , Tyr, (C)Arg, Lys(cpd), Orn(cpd) , Dbu(cpd) , Dpr(cpd), (A)Phe, (3I)Tyr or His; AA 6 is U * , 3-D-NAL, (B)D-Trp, (A')D-Phe, (D)D-Orn, (D)D-Lys, (D)D-Dbu, (D)D-Dpr, D-Har, D-Tyr, (E)D-His, D-PAL, (C)D-Arg or a suitable lipophilic D-isomer; A' is A, NH 2 , NHCH 3 or gua; C is H or lower alkyl; D is G, cpd or an aryl group; E is H, imBzl or dinitrophenol; AA 7 is Nle, Leu, NML, (A)Phe, Met, Nva, Tyr, (B)Trp or PAL; AA 8 is U * , (C')Arg, (C)Har or ILyε; C » is H or di-lower alkyl; AA 10 is D-Ala-NH 2 , Gly-NH 2 , AzaGly-NH 2 or NH(R); R is lower alkyl, preferably CH 2 CH 3 ; and U * is as defined above. When AA 1 is /3-D-NAL and AA 5 is not Arg, then AA 6 is preferably U * , 4-NH 2 -D-Phe, D-Lys, D-Orn, D-Har, D-His, 4-gua-D-Phe, D-PAL or D-Arg. By dehydroPro is meant 3,4 dehydroproline, C 5 H 7 0 2 N. By /3-D-NAL is meant the D-isomer of alanine which is substituted by naphthyl on the 3-carbon atom, i.e., also 3-D-NAL. Preferably 3-D-2NAL is employed wherein the attachment to naphthalene is at the 2-position on the ring structure; however, /3-D-1NAL may also be used. The preferred 1-position residues are /3-D- NAL, substituted D-Phe and optionally substituted D-Trp. PAL represents alanine which is substituted by pyridyl on the / 3-carbon atom; preferably the linkage is to the 3-position on the pyridine ring. When U * is not in the 5- position, it is preferably Tyr, Arg or Lys(cpd). By NML is meant N°CH 3 -L-Leu. By Dbu is meant alpha, gamma diamino butyric acid, and by Dpr is meant a , β diamino propionic acid. By Aph is meant NH 2 Phe which should be assumed to be 4- or para-a ino Phe. By 4-gua-D-Phe is meant a residue of D-Phe having guanidine substituted in the para-position. By AzaGly-NH 2 is meant NHNHC0NH 2 . The guanidino group of an Arg residue in the 5- or 6-position may be substituted by lower alkyl, i.e. 1 to 4 carbon atoms, e.g., propyl(Pr). When D-Lys, D-Dbu, D-Dpr or D-Orn is present in the 6-position and it is not a part of an unusual amino acid U * , its side-chain-amino group may be acylated by an acyl group which may be aliphatic, heterocyclic or aromatic, e.g. nicotinic acid, or may be substituted by an aryl group having not more than 1 phenyl ring. When U * is not present in the 6-position, it is preferably D-PAL or D-Lys(cpd). The 7-position residue is preferably Leu, NML, Nle or Phe. If the 8- position residue is not U * , it is preferably ILys. Biopotent GnRH agonists of the invention are represented by the following Formula (F 2 ) : pGlu-Hiε-Trp-Ser-Tyr-U * -AA 7 -Arg-Pro-AA 10 , wherein AA 7 and AA 10 are as defined hereinbefore; preferably AA 7 is Leu or NML and AA 10 is NHCH 2 CH 3 . A preferred subgenus of protected amino acids which are synthesized has the formula: u IN— -r HO-C-CH-(CH,) .- ( ) )-N-C-NΪ (X 1 )NH H R 2 or where j is an integer from 1 to 3; R 2 is H, alkyl, alkenyl, alkynyl, aryl, OH, NH 2 or heterocycle such as methylpyridyl, pyri idinyl or purinyl; and X 1 is an α- amino protecting group. One preferred method is for the synthesis of a subgenus of protected amino acids having the following formula which are suitable for making GnRH antagonists and other peptides: Another such subgenus of amino acids prepared by the method has the formula: where j is 1, 2 or 3; X 1 is an α-amino protecting group and R 2 is lower alkyl (C 1 -C 6 ) , cyclohexyl, phenyl, pyridyl, methylpyridyl or histaminyl; or Yet another preferred subgenus of amino acids which are synthesized have the formulas: or (b) where R 2 is lower alkyl, pyridyl or methylpyridyl and X 1 is an α-amino protecting group, preferably Boc. The protected amino acids made by the present invention can be used to synthesize peptides by classical solution chain elongation synthesis or by a solid phase technique. A chloromethylated resin or a hydroxy ethylated resin may be used; however, a methylbenzhydrylamine(MBHA) resin, a benzhydrylamine (BHA) resin or some other suitable resin known in the art which directly provides a C-terminal amide or substituted amide upon cleavage is preferably employed when such a C- terminus is desired. For example, peptides having a substituted amide at the C-terminus are preferably synthesized using an N-alkylamino methyl resin as taught in United States Patent No. 4,569,967, issued February 11, 1986. Solid phase synthesis is conducted in a manner to stepwise add amino acids in the chain in the manner set forth in detail in the U.S. Patent No. 4,211,693. Side-chain protecting groups, as are well known in the art, are preferably included as a part of any amino acid which has a particularly reactive side chain and optionally in the case of others, such as Trp, which amino acids are to be coupled in the chain being built upon the resin. Such synthesis provides the fully protected intermediate peptidoresin. Chemical intermediates for GnRH antagonists having an unnatural amino acid in the 5- and the 6- positions, which are made by solution or solid-phase synthesis are represented by the formula: X 1 -AA-AA 2 (X 5 )-AA 3 (X 2 )-Ser(X 3 )-U * -U * -AA 7 (X 2 or X 7 )-AA 8 (X 5 or X 6 )-Pro-X 8 wherein: U * is as defined hereinbefore. X 1 is an α-amino protecting group of the type known to be useful in the art in the stepwise synthesis of polypeptides and when G in the desired peptide composition is a particular acyl group, that group may be used as the protecting group. Among the classes of α-amino protecting groups covered by X 1 are (1) acyl-type protecting groups, such as formyl(For), trifluoroacetyl, phthalyl, p-toluenesulfonyl(Tos) , benzoyl(Bz), benzenesulfonyl, dithiasuccinoyl(Dts) , o-nitrophenylsulfenyl(Nps) , tritylsulfenyl, o-nitrophenoxyacetyl, acrylyl(Acr) , chloroacetyl, acetyl(Ac) and γ-chlorobutyryl; (2) aromatic urethan-type protecting groups, e.g., benzyloxycarbonyl(Z) , fluorenylmethyloxycarbonyl(F oc) and substituted benzyloxycarbonyl, such as p-chlorobenzyloxy- carbonyl(C1Z) , p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl and p-methoxybenzyloxycarbonyl; (3) aliphatic urethan protecting groups, such as tertbutyloxycarbonyl(Boc) , diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl and allyloxycarbonyl; (4) cycloalkyl urethan-type protecting groups, such as cyclopentyloxycarbonyl, adamantyloxycarbonyl and cyclohexyloxycarbonyl; (5) thiourethan-type protecting groups, such as phenylthiocarbonyl; (6) alkyl-type protecting groups, such as allyl(Aly), triphenylmethyl(trityl) and benzyl(Bzl); (7) trialkylsilane groups, such as trimethylsilane. The preferred α-amino protecting group is Boc. X 2 is hydrogen or a protecting group for the indole nitrogen of Trp, such as Bz, Ac or For. In many syntheses there is no need to protect Trp, and such protection is not used if acylated D-Trp is present elsewhere in the peptide. X 3 is a protecting group for the hydroxyl side chain of Ser or Thr, e.g. Ac, Bz, trityl, DCB or benzyl ether(Bzl) and is preferably Bzl. X 4 is hydrogen or a protecting group for the phenolic hydroxyl group of Tyr selected from the group consisting of tetrahydropyranyl, tert-butyl, trityl, benzyl, Z, 2-bromobenzyloxycarbonyl(2BrZ) and 2,6-dichlorobenzyl(DCB) . 2BrZ is preferred. X 5 is a protecting group for a side chain guanidino group, such as that in Arg or Har, or for the imidazole group of His, such as nitro, Tos, trityl, adamantyloxycarbonyl, Z and 2,4-dinitrophenol(Dnp) , or X 5 may be hydrogen, which means there is no protection on the side chain group atoms. Tos is generally preferred. X 6 is a protecting group for an amino side chain group, primary or secondary amino, such as Z or 2C1Z. X 7 is hydrogen or a protecting group for Met, such as oxygen; Met is generally left unprotected. X 8 may be Gly-NH-[resin support], D-Ala-NH- [resin support] or N(A)-[resin support ' ]; X 8 may also be an amide or an ester protecting group or the like (when solution synthesis is used) either of Gly or of D-Ala or a substituted amide attached directly to Pro or NHNHCONH 2 . The criterion for selecting side chain protecting groups for X 2 -X 7 is that the protecting group should be stable to the reagent under the reaction conditions selected for removing the α-amino protecting group (preferably Boc) at each step of the synthesis. Protecting groups generally should not be split off under coupling conditions but should be removable upon completion of the synthesis of the desired amino acid sequence under reaction conditions that will not alter the peptide chain. When the X 8 group is Gly-NH-[resin support] or D-Ala-NH-[resin support], an amide bond connects Gly or D-Ala to a BHA resin or to a MBHA resin. When the X 8 group is N(A)-[resin support], a substituted amide bond connects Pro to an N-alkylaminomethyl (NAAM) resin. When X 8 is AzaGly-NH 2 , the peptide is preferably made by classical solution synthesis, as disclosed in U.S. Patent No. 4,234,571. When G is acetyl, for example, in the final formula, a reaction is preferably carried out with the peptide on the resin (after deblocking the α-amino group while the side-chain groups remain protected) , e.g. by reacting with acetic acid in the presence of diisopropyl or dicyclohexyl carbodiimide (DIC or DCC) or preferably with acetic anhydride. Other suitable reactions as known in the art can also be used. Purification of the peptide is effected by ion exchange chromatography on a CMC column, followed by partition chromatography using the elution system: n-butanol;0.IN acetic acid (1:1 volume ratio) on a column packed with Sephadex G-25, or by using HPLC, as known in the art and specifically set forth in J. Rivier, et al. J. Chromatoσraphv. 288 (1984) 303-328. The GnRH antagonists are effective at levels of less than 100 micrograms per kilogram of body weight, when administered subcutaneously at about noon on the day of proestrus, to prevent ovulation in female rats. For prolonged suppression of ovulation, it may be necessary to use dosage levels in the range of from about 0.1 to about 2.5 milligrams per kilogram of body weight. These analogs are particularly soluble at physiological pHs and thus can be prepared as relatively concentrated solutions for administration. These antagonists can also be used to regulate the production of gonadotropins and sex steroids for other purposes as indicated hereinbefore. In the following formulas, the U * residues are defined in terms of the original amino acid residue having a side chain amino group plus the modification in question which is set forth in the accompanying parentheses. The original residue is either aminophenylalanine (D- or L-Aph) where the amino substitution is in the 4- or para-position on the ring, or aminohomoPhe (Hph) or aminohomohomoPhe. It is incorporated in the growing peptide chain by adding the suitably protected unnatural amino acid U * as a part of the usual chain elongation process. With respect to the modified side chain amino groups, the following abbreviations are used in the Examples which follow: act=3-acetylamino 1,2,4 triazole atz=3-amino 1,2,4 triazole bcg=aminobutyl cyanoguanidino bzcg=aminobenzyl cyanoguanidino chcg=aminocyclohexyl cyanoguanidino ecg=aminoethyl cyanoguanidino icg=aminoisopropyl cyanoguanidino hcg=aminohexyl cyanoguanidino hicg=histaminyl cyanoguanidino (ethylimidazole) mcg=aminomethyl cyanoguanidino ncg=aminoethyl(1 or 2)naphthyl cyanoguanidino mncg=aminomethyl(1 or 2)naphthyl cyanoguanidino pcg=aminopropyl cyanoguanidino trcg=indole ethylamino cyanoguanidino(tryptamino cyanoguanidino) mpcg=aminomethyl pyridyl cyanoguanidino (number indicates position of aminomethyl group on pyridyl ring) EXAMPLE 1 Preparation of D and L N α -Boc-4-f5 • - (3 '-amino-lH-1' . 4'-triazolyl.. -Aminophenylalanines from D and L phenylalanine Preparation of the amino acid 3-(4- nitrophenyl)alanine has frequently utilized various means of aromatic ring nitration on phenylalanine. The nitration procedure most frequently used employs a solution of nitric acid dissolved in sulfuric acid and is commonly referred to as a "mixed acid". Contrary to the high yields often reported, it was later found that actual nitration occurs in all possible locations of the aromatic portion of the amino acid (ortho, meta and para) , and modification of the reaction conditions only brought about minor changes in the isomeric ratios. Repeated crystallizations from boiling water could remove the undesired o- and m-isomers, but the yields were only about 25% of the theoretical. Although other nitration compositions consisting essentially of nitric acid and acetic anhydride or nitrate salts in various acids have been used as well, there appeared to be no advantage to these variations of nitration because yields were modest and the isomeric distributions were not reported. Of the various methods cited in the literature, one factor of the reaction remains constant, i.e. the ratio of the nitrating agent to the quantity of Phe used as a reactant. The value is generally 1.1 to 1.3 equivalents of appropriate nitrate to avoid excess nitration. Excess sulfuric acid is generally added to the reaction to help in the formation of the nitration agent (NO* 2 ) because nitrations are thought to be most rapid in systems which are above 90% sulfuric acid. It has now been found that the use of larger amounts of sulfuric acid may lead to a yellow amino acid during the isolation; however, by greatly increasing the amount of nitric acid so as to provide at least about 2 moles (equivalents) of HN0 3 per mole of amino acid, and preferably between about 2 and about 3.5 equivalents, a cleaner product is produced. Preferably, sulfuric acid is provided in an amount of between about 2 and about 3 equivalents per equivalent of nitric acid. Moreover, duration of action in conjugation gave higher yields. After recrystallization from water, the 3-(4- nitrophenyl)alanine was checked for potential contamination of unwanted meta- or ortho-substituted isomers by use of CZE, and under conditions known to elute the other isomers, only the para-substituted isomer was seen. The α-amino group is then protected with an aliphatic urethane protecting group, preferably Boc. It is then shown that both D- and L-Boc-4- Aph(atz) amino acids can be readily prepared from the corresponding Boc-4-Aph amino acids by reaction of the para-amino function with diphenylcyanocarbonimidate. followed by treatment with hydrazine hydrate. This two- step reaction is essentially a one-pot procedure, and although a concentration/solvent exchange is carried out between the two steps, such is unnecessary if the reaction is done in an alcoholic solvent, such as isopropanol. The limited solubility of Boc-4-Aph(atz) in dichloromethane is overcome by using N-methylpyrrolidine (NMP) as a co-solvent. The experimental conditions set forth hereinafter are illustrative of the preparation of these amino acids. 4-Nitrophenyl-D-Alanine: Into a 600 mL beaker equipped with a stirring bar and maintained in an ice bath, cold, concentrated (18 molar) sulfuric acid (200 mL) was added slowly to 150 mL of cold, concentrated (16 molar) nitric acid (about 2.4 mole). After cooling to 3°C, D-phenylalanine (180 g, 1.09 mole) was added in 5-10 gram portions over 1 hour to ensure that the temperature stayed about 5°C. The reaction was stirred at ice bath temperature (2-5°C) for 4 hours. The reaction was then quenched by pouring onto 3 kg of ice, followed by cautious neutralization of the reaction with concentrated ammonium hydroxide. At pH 5-6, the solution became turbid, and 4-nitrophenyl-D-alanine precipitated. The precipitate was collected by filtration and washed with ice water (1L) . This amino acid was then twice recrystallized from 700 mL of hot water. The recrystallized product was collected and dried under high vacuum to give 157 grams of 4-nitrophenyl-D-alanine (0.75 mole; 68%). [o] 0 = -6.9° (c = 0.86 in IN HC1) ; mp = 238- 244°C. 4-Nitrophenyl-L-Alanine: The reaction described above for 4-nitrophenyl-D-alanine was repeated for the L isomer. About 125 grams of L-Phe was nitrated as described above and resulted in about 124 grams of 4- nitrophenyl-L-alanine (about 77% yield). [α] 0 = +6.8° (c = 1.0 in IN HC1) . N"-Boc-4-Nitrophenyl-D-Alanine: 4-Nitrophenyl- D-alanine (157 grams, 0.75 mole) was dissolved in 500 mL of 40% aqueous tert-butyl alcohol, and the pH was adjusted to 9.9 with 2N NaOH. Di-tert-butyldicarbonate (198 g, 0.9 mole) was added over 35 minutes to the stirred reaction, and the mixture was then allowed to stir at ambient temperature for 2 hours. The pH was maintained at about 9.8 over the course of the reaction. The reaction product was then first extracted with petroleum ether (2 x 800 mL) and diethyl ether (1 x 500 mL) , followed by cautious acidification of the aqueous phase to pH 2 with NaHSO^. The aqueous layer was extracted with ethyl acetate; this was then dried over MgS0 4 and concentrated under vacuum to yield a viscous oil that solidified upon trituration with petroleum ether. The Boc-amino acid was collected by filtration, washed with petroleum ether and dried to give 210 g of N α -Boc-4- nitrophenyl-D-alanine (0.68 mole, 90%). [α] D = -7.0° (c = 0.9 in MeOH) ; mp = 100-104°C. N a -Boc-4-Nitrophenyl-L-Alanine: The reaction described above for N α -Boc-4-Nitrophenyl-L-alanine was repeated for the L-isomer. About 123 grams of 4- nitrophenyl-L-alanine was reacted as described and resulted in about 155 grams of N a -Boc-4-Nitrophenyl-L- alanine (about 86% yield). [α] D = +7.1° (c = 1.0 in MeOH) . N a -Boc-4-Aminophenyl-D-Alanine: N β -Boc-4- Nitrophenyl-D-alanine (209 grams, 0.68 mole) was dissolved in 500 mL of ethyl alcohol and acidified with acetic acid (10 mL) . To this was added 0.65 g of 10% Pd/carbon, and the mixture was hydrogenated at about room te perature using H 2 at about 40 psi until the uptake of hydrogen had ceased (6 hours) . The catalyst was removed by filtration, and the reaction products were concentrated under vacuum to yield a viscous oil that solidified upon trituration with petroleum ether. The amino acid was collected by filtration, washed with petroleum ether and dried to give 184 g of N a -Boc-4- aminophenyl-D-alanine (0.659 mole, 97%) having [α] D = - 40.2° (c = 1.0 in EtOAc) and -23.4° (c = 1.0 in MeOH); mp = 128-133°C. N tt -Boc-4-Aminophenyl-L-Alanine: The reaction described above for N a -Boc-4-aminophenyl-D-alanine was repeated for the L-isomer. About 153 grams of N a -Boc-4- nitrophenyl-L-alanine was reacted as described above and resulted in about 134 grams of N a -Boc-4-aminophenyl-L- alanine (about 96% yield) [α] D - +23.6° (c = 1.0 in MeOH). N a -Boc-4-f5 l -f3 , -amino-lH-l t . 2'. 4'- triazolyl..-Aminophenyl-L-Alanine fBoc-L-4Aph(atz. ] : To 9 grams of Boc-L-4-aminophenylalanine (32 mmol) in a 250 L round bottom flask equipped with a mechanical stirrer was added 100 L of dichloromethane and enough N- methylpyrrolidone until all the solid had dissolved (10 mL) . At such time, 8.7 grams (36.6 mmol) of diphenyl- cyanocarbonimidate was added, and the reaction mixture was stirred at ambient temperature for about 17 hours. The reaction mixture was then concentrated under vacuum, and the oil obtained containing the cyanoguanidino intermediate was dissolved in 125 mL of methanol. 15 mL of hydrazine hydrate was added, and the reaction mixture was stirred at ambient temperature for 24 hours. This reaction mixture was then concentrated under vacuum to approximately 50 mL, and the oil obtained was diluted into 350 mL of cold water. Dilute sulfuric acid was added until the pH was about 7, and the aqueous layer was extracted with ether (1 x 150 L) to remove the phenol formed in the reaction. The pH of the aqueous layer was then adjusted to 2-3, at which time a precipitate formed. The reaction product was then extracted with hot ethyl acetate (3 x 250 mL) , and the organic layer dried over MgS0 4 . The organic layer was then concentrated to give an oil which, upon washing with ether, solidified. The powder obtained was filtered, washed with petroleum ether, and then dried to give 9.2 g of Boc-L-Aph(atz) as a tan powder (25.3 mmol, 79% yield) which is also referred to as Boc-Aph(3-amino, 1,2,4 triazole). [α] D = -16.4° (c = 1.0 in MeOH). N a -Boc-4-f5 t -r3'-amino-lH-l' . 2'. 4 1 - triazolyl. )-aminophenyl-D-alanine [Boc-D-4Aphfatz) ] : The reaction described above for the Boc-L-Aph(atz) was repeated for the D-isomer. 10.1 grams of Boc-D-Aph was treated with 10.8 grams of diphenylcyanocarbonimidate followed by 20 mL of hydrazine hydrate; it eventually resulted in 7.8 grams of Boc-D-Aph(atz) (61% yield), having an [α] D = -15.6° (c = 1.0 in MeOH). EXAMPLE 2 The decapeptide [Ac-;0-D-2NAL 1 , (4C1 ) D-Phe 2 , D- 3PAL 3 , Aph (atz ) 5 , D-Aph (atz ) 6 , ILys 8 , D-Ala 10 ] -GnRH is synthesized by solid-phase synthesis. This peptide has the following formula: AC-/3-D-2NAL-(4Cl) D-Phe-D-3PAL-Ser-Aph(3-amino, 1,2,4 triazole)-D-Aph(3-amino, 1,2,4 triazole)-Leu- Lys(isopropyl)-Pro-D-Ala-NH 2 . MBHA resin (0.76 mM/g) is used, and Boc- protected D-Ala is coupled to the resin over a 2-hour period in CH 2 Cl 2 using about a two-fold excess of Boc derivative and DCC as an activating reagent. The D-Ala residue attaches to the MBHA residue by an amide bond. Beginning with about 0.9 grams of resin and using an automated machine, coupling of each amino acid residue, washing, deblocking and the coupling of the next amino acid residue is carried out in accordance with the following schedule: The above schedule is used for coupling of each of the amino acids of the peptide of the invention after the first amino acid has been attached. N a Boc protection is used for each of the remaining amino acids throughout the synthesis. N a Boc-/3-D-2NAL is prepared by a method known in the art, e.g. as described in detail in U.S. Patent No. 4,234,571, issued November 18, 1980; it is also commercially available from SyntheTech, Oregon, U.S.A. Bzl(benzyl ether) is used as a side chain protecting group for the hydroxyl group of Ser. Boc-Lys(Ipr,Z) is used for the 8-position. The side chain groups of 4Aph(atz) in the 5-position and D-4Aph(atz) in the 6-position need not be protected. After deblocking the α-amino group at the N- terminus using trifluoroacetic acid (TFA) , acetylation is achieved using a large excess of acetic anhydride in dichloro ethane. Following completion of the assembly of the peptide and acetylation of the N-terminus, about 1.67 grams of the following intermediate are present: AC-/3-D-2NAL- (4Cl)D-Phe-D-3PAL-Ser(Bzl)-Aph(atz)-D-Aph(atz)-Leu- Lys(Ipr,Z)-Pro-D-Ala-NH-[MBHA resin support]. The peptidoresin is dried, and then cleavage of the peptide from the resin and deprotection of the Ser and the Lys side chains is carried out at 0°C. with HF. Anisole is added as a scavenger prior to HF treatment. After the removal of HF under vacuum, the resin is washed twice with 100 ml. of ethyl ether. The cleaved peptide is extracted from the resin with equal parts of CH 3 CN and H 2 0, repeating the process and using 150 ml. each time. The extracts are pooled and lyophilized, and they provide about 650 mg of a crude peptide powder. Purification of the peptide is then effected by preparative high performance liquid chromatography (HPLC) , as known in the art and specifically set forth in J. Rivier, et al. J. Chromatography. 288, 303-328 (1984). The first preparative RP-HPLC separation uses a TEAP (triethylammonium phosphate) buffer system. This separation is repeated using the same buffer system with a slightly different gradient, and the final separation is carried out using a 0.1% TFA (trifluoroacetic acid) gradient, all as described in detail in the Jj. Chromatography article. About 80.7 milligrams of the decapeptide are obtained, which is referred to as Peptide No. 1. The peptide is judged to be homogeneous using capillary zone electrophoresis (CZE) , as well as by using reversed-phase high performance liquid chromatography and an aqueous triethylammonium phosphate buffer plus acetonitrile. The purity is estimated to be about 97%. Amino acid analysis of the resultant, purified peptide is consistent with the formula for the prepared structure, showing substantially integer-values for each amino acid in the chain; mass spectral analysis is also consistent. The optical rotation is measured on a photoelectric polarimeter as [α] 20 = -2.8+0.5(c=l, 50% acetic acid). Peptide No. 1 is assayed in vivo and also tested in vitro. In vitro testing is carried out using dissociated rat pituitary cells maintained in culture for 4 days prior to the assay. The levels of LH mediated in response to the application of peptides is assayed by specific radioimmunoassay for rat LH. Control dishes of cells only receive a measure which is 3 nanomolar in GnRH; experimental dishes receive a measure 3 nanomolar in GnRH plus a measure having either the present standard antagonist for comparison purposes i.e. [Ac-dehydro Pro 1 , (4F)D-Phe 2 , D-Trp 3,6 ]-GnRH or the test peptide, in concentrations ranging from 0.01 to 10 nanomolar. The amount of LH secreted in the samples treated only with GnRH is compared with that secreted by the samples treated with the peptide plus GnRH. The ability of the test peptide to reduce the amount of LH released by 3 nanomolar GnRH is compared to that of the present standard peptide. The in vivo testing determines effectiveness to prevent ovulation in female rats. In this test, a specified number of mature female Sprague-Dawley rats, e.g. five to ten, each having a body weight from 225 to 250 grams, is injected with a specified microgram dosage of peptide in either saline, bacteriostatic water, polyethylene glycol, corn oil or mixtures of the above with ethanol at about noon on the day of proestrus. Proestrus is the afternoon of ovulation. A separate female rat group is used as a control to which the peptide is not administered. Each of the control female rats ovulates on the evening of proestrus; of the rats treated, the number of them which ovulate is recorded. Each of the peptides is considered to be totally effective to prevent ovulation of female rats at a dose of about 500 micrograms. The peptide referred to as Peptide No. 1 is effective to block GnRH-induced LH secretion in vitro at low concentrations. The peptide is considered to be effective to prevent ovulation of female mammals at low dosages. The following Table A shows the results of in vivo testing of this GnRH antagonist with the dosages being given in micrograms: Additional peptides, all of which include at least one D-isomer unnatural amino acid residue, are set forth hereinafter. EXAMPLE 3 Peptides as indicated in TABLE B having the formula: AC-3-D-2NAL-(4C1)D-Phe-D-3PAL-Ser-AA 5 -AA 6 -Leu-AA 8 - Pro-D-Ala-NH 2 are prepared by the solid-phase procedure referred to in Example 2. The unnatural amino acids are synthesized using the process of Example 1 but substituting the appropriate reagent for hydrazine. For example, Peptide No. 11 is synthesized using a protected amino acid prepared by treating the cyanoguanidino intermediate with isopropylamine in DMF for 24 hours at room temperature. TABLE B All peptides listed in Table B are considered effective to block GnRH-induced LH secretion in vitro at some reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages. EXAMPLE 4 Peptides as indicated in TABLE C having the formula: AC-3-D-2NAL-(4C1)D-Phe-D-3PAL-Ser-AA 5 -AA 6 -AA 7 - ILys-Pro-AA 10 are prepared by the solid-phase procedure referred to above. TABLE C The peptides listed in Table C are considered effective to block GnRH-induced LH secretion m vitro at a reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages. EXAMPLE 5 Peptides as indicated in TABLE D having the formula: 'pGlu-His-Trp-Ser-Arg-AA 6 -AA 7 -Arg-Pro-AA 10 are prepared by the solid phase procedure referred to above. TABLE D The peptides described in TABLE D are considered to be effective to cause the release of LH and FSH in female rats. All of them are considered to be substantially more effective than native GnRH. The peptides of the invention are often administered in the form of pharmaceutically acceptable, nontoxic salts, such as acid addition salts, or of metal complexes, as is well known in this art. For example, an aqueous solution of the peptide can be repeatedly treated with IN acetic acid and then lyophilized to yield the acetic acid salt thereof. The pharmaceutical compositions will usually contain the peptide in conjunction with a conventional, pharmaceutically-acceptable carrier. Usually, the dosage will be from about 10 micrograms to about 0.5 milligram of the peptide per kilogram of the body weight of the host when given intravenously, intramuscularly or subcutaneously. Overall, treatment of subjects with these peptides is generally carried out in the same manner as the clinical treatment using other antagonists or agonists of GnRH using a suitable carrier in which the peptide is soluble. Although the invention has been described with regard to its preferred embodiments, it should be understood that changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims which are appended hereto. In the amino acids, instead of making the modifications upon an a ino-substituted Phe either ho oPhe or homohomoPhe having the appropriate amino substitution can be used to create unnatural amino acids having similar properties in either the D- or L-isomer forms. Particular features of the invention are emphasized in the claims which follow.

Claims: CLAIMS : 1. A method of making an unnatural amino acid useful for synthesis of a peptide by a chain elongation process, which method comprises subjecting an α-amino acid having the formula: \0/ ( CH 2 ) j -CHNH 2 COOH, wherein j is 1, 2 or 3, to nitration conditions by forming a reaction mixture which includes said amino acid, concentrated sulfuric acid and concentrated nitric acid having at least about 2 moles of HN0 3 for each mole of α-amino acid and maintaining said reaction mixture at a temperature of about 5°C or below to cause said nitration to occur predominantly at the 4-position, reacting said 4N0 2 -substituted product with an appropriate reagent to add an amino-protecting group to said α-amino acid, treating said protected amino acid to hydrogenate said substituted nitro moiety and transform it to an amino moiety, and dissolving said hydrogenated amino acid in a suitable solvent and reacting it with diphenylcyano- carbonimidate to form a cyanoguanidino intermediate which is reactive with a nucleophilic moiety to produce a desired unnatural amino acid. 2. A method according to Claim 1 wherein concentrated nitric acid is employed in said nitrating step in an amount of between about 2 and about 3.5 moles of HN0 3 per mole of α-amino acid. 3. A method according to Claim 2 wherein said concentrated sulfuric acid is employed in an amount of about 2 to about 3 equivalents per each equivalent of HN0 3 . 4. A method according to Claim 1 wherein said 4N0 2 -substituted product is reacted with a reagent that results in the protection of the α-amino group by an aliphatic urethane protecting group. 5. A method according to Claim 4 wherein said protecting group is tert-butyloxycarbonyl(Boc) . 6. A method according to Claim 5 wherein said reagent is di-tert-butyldicarbonate. 7. A method according to Claim 1 wherein said protected α-amino acid is dissolved in an alcohol and hydrogenated using a palladium/carbon catalyst. 8. A method according to Claim 1 wherein said hydrogenated compound is dissolved in a mixture of dichloromethane and N-methylpyrrolidine for said reaction to form said cyanoguanidino intermediate. 9. A method according to Claim 8 wherein hydrazine in the form of hydrazine hydrate is added to a solution in which said intermediate is dissolved. 10. A method according to Claim 9 wherein j is 1. 11. A method according to any one of Claims l to 10 wherein said intermediate is reacted with hydrazine to create a 3-amino,l,2,4 triazole moiety attached to the 4-position of the phenyl ring of the side chain of said amino acid. 12. A method of synthesizing a peptide by chain elongation from the C-terminus thereof, which method comprises deprotecting the α-amino group of a peptide intermediate and reacting same to form an amide bond with an α-amino acid made by the method of Claim 1. 13. An amino acid made by the method of Claim 1, which amino acid has the formula: or NH 2 / wherein X, is an α-amino-protecting group, j is 1, 2 or 3, and R 2 is alkyl, modified alkyl, alkenyl, alkynyl, aryl or methylpyridyl.

Inventors: HOEGER CARL A, PORTER JOHN S, RIVIER JEAN E F, HOEGER, CARL, A., PORTER, JOHN, S., RIVIER, JEAN, E., F.
Applicants: SALK INST FOR BIOLOGICAL STUDI, THE SALK INSTITUTE FOR BIOLOGICAL STUDIES
Application year: 1994
Main classification: C07C277
IPC codes: A61K38, C07C277, C07C279, C07D249, C07K7